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Frontiers in Cell and Developmental... 2023Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that... (Review)
Review
Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle.
PubMed: 36994100
DOI: 10.3389/fcell.2023.1139367 -
Molecular Biology of the Cell May 2023The human centromere comprises large arrays of repetitive α-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are...
The human centromere comprises large arrays of repetitive α-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Because CENP-A is bound to only a fraction of the α-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of α-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the centromere chromosome orientation fluorescence in situ hybridization (Cen-CO-FISH) technique together with structured illumination microscopy. Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that α-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in the presence or absence of spindle's microtubules. Using expansion microscopy, we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of α-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Collectively, our data provide a 3D reconstruction of α-satellite DNA along with CENP-A clusters that outlines the overall architecture of the mitotic centromere.
Topics: Humans; DNA, Satellite; Centromere Protein A; Microscopy; In Situ Hybridization, Fluorescence; Chromosomal Proteins, Non-Histone; Autoantigens; Centromere; DNA
PubMed: 36947236
DOI: 10.1091/mbc.E22-08-0332 -
Frontiers in Cell and Developmental... 2023was originally identified as a part of an aberrant fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the fusion gene in...
was originally identified as a part of an aberrant fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the fusion gene in the tumor genome, the cell loses one wild type allele. Our previous study demonstrated that the loss of (homologue of human ) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both genes of DLD-1 cell were tagged with at its 5'-end using a CRISPR/Cas9 system, treatment of the () DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of and (a mutant that has low affinity for Aurora B) in the () DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.
PubMed: 36875767
DOI: 10.3389/fcell.2023.987153 -
Journal of Genetics 2023The nine Blume populations studied from Pir Panjal contained 20 chromosomes. This count is not reported so far in Indian populations. Currently, comparison of tapetal...
The nine Blume populations studied from Pir Panjal contained 20 chromosomes. This count is not reported so far in Indian populations. Currently, comparison of tapetal and meiotic cells revealed the existence of synchrony in different developmental phases. Young tapetal cells at prometaphase co-occurred with the pollen mother cells (PMCs) at diakinesis to metaphase, mature tapetal cells with disintegrated chromatin material co-occurred with tetrads and no tapetal cells were found at mature pollen stage. Cytological studies in young tapetal cells revealed most of these to be endopolyploid, with each having 40 chromosomes. While outnumbering somatic cells contained clear 40 chromosomes which seemed to be the outcome of endomitosis, a sizeable number of cells possessed 40 sticky chromosomes at metaphase. Later chromosomes are likely to form restitution nucleus. Mature tapetal cells, occurring singly/cytomictically connected (3.2-26.31%) or showing coalescence (10.5-22.8%), did not contain recognizable chromosomes. Instead, they were characterized by disintegrated nuclear content. Further, meiotic studies revealed that the present population contained all/outnumbering euploid cells (2=20); many of which exhibited nearly regular behaviour. However, 6.5-26.9% meiocytes of eight populations and 47% cells of P-Khe population depicted aneuploidy/contained quadri-octavalents, with per cent pollen viabilities of these ranging from 38.6 to 49.9. Going by the normal tapetal development in , existence of various chromosomal anomalies seems to have accounted for the reduction in gametic fertility of this taxon.
Topics: Viola; Metaphase; Chromosome Disorders; Diploidy; Reproduction; Meiosis
PubMed: 36823681
DOI: No ID Found -
Molecular Biology of the Cell May 2023The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to...
The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to address two questions: how rapidly the microtubule-kinetochore connections are made, and how accurate these connections are. In most previous numerical simulations, the model geometry was drastically simplified. Using the computational platform, we previously introduced a geometrically and mechanically realistic 3D model of the prometaphase mitotic spindle, and used it to evaluate thermal noise and microtubule kinetics effects on the capture of a single chromosome. Here, we systematically investigate how geometry and mechanics affect a spindle assembly's speed and accuracy, including nuanced distinctions between , , and chromosomes. We find that softening of the centromere spring improves accuracy for short chromosome arms, but accuracy disappears for long chromosome arms. Initial proximity of chromosomes to one spindle pole makes assembly accuracy worse, while initial chromosome orientation matters less. Chromokinesins, added onto flexible chromosome arms, allow modeling of the polar ejection force, improving a spindle assembly's accuracy for a single chromosome. However, spindle space crowding by multiple chromosomes worsens assembly accuracy. Our simulations suggest that the complex microtubule network of the early spindle is key to rapid and accurate assembly.
Topics: Chromosomes; Centromere; Spindle Apparatus; Kinetochores; Microtubules; Prometaphase; Chromosome Segregation; Mitosis
PubMed: 36790911
DOI: 10.1091/mbc.E22-11-0507 -
Cells Jan 2023Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was...
Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.
Topics: Humans; Centromere; Ligases; Mitosis; Molecular Chaperones; Nuclear Pore Complex Proteins; Protein Inhibitors of Activated STAT; Sumoylation
PubMed: 36766713
DOI: 10.3390/cells12030372 -
Frontiers in Cell and Developmental... 2022PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the...
PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.
PubMed: 36733339
DOI: 10.3389/fcell.2022.1069946 -
BioRxiv : the Preprint Server For... Jan 2023Tumor cell lines with elevated chromosome numbers frequently have correlated elevations of Mps1 expression and these tumors are more dependent on Mps1 activity for their...
Tumor cell lines with elevated chromosome numbers frequently have correlated elevations of Mps1 expression and these tumors are more dependent on Mps1 activity for their survival than control cell lines. Mps1 is a conserved kinase involved in controlling aspects of chromosome segregation in mitosis and meiosis. The mechanistic explanation for the Mps1-addiction of aneuploid cells is unknown. To address this question, we explored Mps1-dependence in yeast cells with increased sets of chromosomes. These experiments revealed that in yeast, increasing ploidy leads to delays and failures in orienting chromosomes on the mitotic spindle. Yeast cells with elevated numbers of chromosomes proved vulnerable to reductions of Mps1 activity. Cells with reduced Mps1 activity exhibit an extended prometaphase with longer spindles and delays in orienting the chromosomes. One known role of Mps1 is in recruiting Bub1 to the kinetochore in meiosis. We found that the Mps1-addiction of polyploid yeast cells is due in part to its role in Bub1 recruitment. Together, the experiments presented here demonstrate that increased ploidy renders cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why hyper-diploid cancer cells exhibit elevated reliance on Mps1 expression for successful chromosome segregation.
PubMed: 36712123
DOI: 10.1101/2023.01.09.523325 -
Science Advances Jan 2023Mitotic perturbations activate the spindle assembly checkpoint (SAC) that keeps cells in prometaphase with high CDK1 activity. Prolonged mitotic arrest is eventually...
Mitotic perturbations activate the spindle assembly checkpoint (SAC) that keeps cells in prometaphase with high CDK1 activity. Prolonged mitotic arrest is eventually bypassed by gradual cyclin B decline followed by slippage of cells into G without chromosome segregation, a process that promotes cell transformation and drug resistance. Hitherto, the cyclin B1 decay is exclusively defined by mechanisms that involve its proteasomal degradation. Here, we report that hyperphosphorylated HIPK2 kinase accumulates in mitotic cells and phosphorylates the Rett syndrome protein MeCP2 at Ser, a regulation that is counteracted by CDC14B phosphatase. MeCP2 phosphorylation leads to the enhanced translation of cyclin B1, which is important for cells with persistent SAC activation to counteract the proteolytic decline of cyclin B1 and therefore to suspend mitotic slippage. Hence, the HIPK2/CDC14B-MeCP2 axis functions as an enhancer of the SAC-induced mitotic block. Collectively, our study revises the prevailing view of how cells confer a sustainable SAC.
Topics: Cell Cycle Checkpoints; Cyclin B1; M Phase Cell Cycle Checkpoints; Mitosis; Phosphorylation; Dual-Specificity Phosphatases; Protein Serine-Threonine Kinases; Carrier Proteins; Methyl-CpG-Binding Protein 2
PubMed: 36662865
DOI: 10.1126/sciadv.add6982 -
PLoS Genetics Jan 2023The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic...
The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.
Topics: Animals; Caenorhabditis elegans; Spindle Apparatus; Microtubules; Spindle Poles; Meiosis; Oocytes
PubMed: 36608115
DOI: 10.1371/journal.pgen.1010363